Pharmaceutical preparation containing protein C and a thrombolytically active substance

ABSTRACT

A pharmaceutical preparation contains protein C and a thrombolytically active substance that does not activate protein C. This preparation prevents reocclusion usually occurring in the course of thrombolysis therapy.

BACKGROUND OF THE INVENTION

The invention relates to a pharmaceutical preparation containing proteinC.

Many surgical procedures increase the risk of venous and arterialthromboses and of thromboembolism. Arterial and venous thromboses alsomay be caused by diseases. However, antithrombotic and thrombolytictherapies involve undesired side effects, such as bleeding orreocclusion.

The thrombolytic activity of substances, such as t-PA, urokinase,streptokinase or plasminogen, is based on the release of plasmin, whichenables the dissolution of thrombi. At the same time, it is observedthat thrombin is generated when administering thrombolytically activesubstances. The thrombin generation and subsequent reocclusion arepresumed to be a generally undesired side effect of successfulthrombolysis (Circulation 83, 937-944, (1991)).

In addition to an elevated thrombin activity, a change of the protein Cconcentration in blood is observed in thrombolysis patients (Seminars inThrombosis and Hemostasis 16, 242-244 (1990)). Therapy is effectedeither with t-PA or with heparin or with a combination of t-PA andheparin. A reduction of the protein C concentration is observed onlywhen administering t-PA-containing preparations. The direct degradationof protein C by t-PA or plasmin has not been considered, however.

In connection with undesired side effects of thrombolysis, it issuggested in EP-A-0 318 201 to use activated protein C (aPC) alone or incombination with a thrombolytic agent to prevent acute arterialthrombotic occlusions, thromboembolism or stenosis. aPC is known to bean anticoagulatively effective enzyme, which, on the one hand, inhibitsthe formation of thrombin due to the proteolytic degradation ofactivated coagulation factor V and activated coagulation factor VIIIand, on the other hand, supports fibrinolysis. For this reason, the doseof thrombolytic agent may be reduced.

Likewise, it is suggested in Blood 74, Suppl. 1, 50a, Abstract 176(1989) to use a combined preparation containing aPC and urokinase toprevent the formation of thrombi in order to reduce the thrombolytic orantithrombotic doses of urokinase and simultaneously combat bleedingcomplications occurring in thrombolysis therapies. However, theadministration of activated protein C suffers the disadvantage that thetendency to bleeding is favored by the immediate anticoagulant effect ofaPC.

SUMMARY OF THE INVENTION

The invention has as its object to eliminate this disadvantage and toprovide a pharmaceutical preparation that prevents reocclusion caused byan elevated thrombin activity after thrombolysis therapy has beencarried out.

The preparation according to the invention contains protein C and athrombolytically active substance that does not activate protein C.

It has been shown that the preparation according to the inventionenables successful thrombolysis therapy without the risk of reocclusion.

Urokinase, tPA, Lys-plasminogen or streptokinase are particularlysuitable as thrombolytically active substances.

Consequently, the invention also relates to the use of athrombolytically active substance unable to activate protein C, incombination with protein C, to produce a drug for the treatment ofthromboses and for the prevention of reocclusion.

The invention is based on the findings that plasmin generated bytherapeutic thrombolytics not only degrades fibrin, but surprisinglyalso degrades protein C at the very high concentrations applied duringthrombolysis therapy, thus provoking protein C deficiency and inducinghypercoagulability. This may be prevented by administering thethrombolytically active substance in combination with protein C or byadministering protein C in the course of thrombolysis therapy, i.e.,prior to, during and/or after thrombolysis therapy.

Preferably, protein C is contained in the preparation according to theinvention at 10 to 50 U/mg protein. When ready for application, itshould contain 90 to 450 U/ml. The content of thrombolytic agent in thepreparation according to the invention appropriately is chosen so thatusual amounts will be administered when applied.

The protein C-containing preparation according to the invention may beadministered as an injection (30 to 80 U/kg) two or three times a day oras a continuous infusion (15 to 30 U/kg/h).

The invention will be described in more detail in the following.

BRIEF DESCRIPTION OF THE FIGURE

FIG. 1 depicts the degradation of protein C by Plasmin.

DETAILED DESCRIPTION OF THE INVENTION

Preparation of Protein C

Highly pure protein C was recovered from a crude protein C fractionobtained from commercially available prothrombin complex concentrate.Purification was effected by affinity chromatography by means ofmonoclonal antibodies. Monoclonal anti-protein C antibodies wereproduced as follows:

BALB/C mice were immunized with 100 μg human protein C byintraperitoneal injection at two-week intervals. After six weeks,another 50 μg of human protein C was injected and fusion was carried outthree days later. The myeloma cell line (P3-X-63-AG8-653, 1.5×10⁷ cells)was mixed with 1.7×10⁸ mouse spleen cells and fused according to themodified method of Köhler & Milstein by using PEG 1500 (Köhler G.,Milstein C., Nature 256 (1975), 495-497).

Positive clones, assayed by means of ELISA, were subcloned twice.Ascites production was effected by injection of 5×10⁶ hybridoma cellsper BALB/C mouse two weeks after Pristan treatment.

The immunoglobulin was purified from ascites by means of ammoniumsulfate precipitation and subsequent chromatography on QAE-Sephadex and,further, by chromatography on Sephadex G200. To reduce the risk oftransmission of murine viruses, the antibody was subjected to a furthervirus inactivation step prior to immobilization. The monoclonal proteinC antibodies thus obtained were coupled to CNBr-activated Sepharose 4B(Pharmacia). The following buffers were used for the purification ofprotein C by means of affinity chromatography:

Adsorption buffer: 20 mM Tris, 2 mM EDTA, 0.25 M NaCl and 5 mMbenzamidine;

Washing buffer: 20 mM Tris, 1 M NaCl, 2 mM benzamidine, 2 mM EDTA, pH7.4;

Elution buffer: 3 M NaSCN, 20 mM Tris, 1 M NaCl, 0.5 mM benzamidine, 2mM EDTA.

In detail: The prothrombin complex concentrate was dissolved in theadsorption buffer, with approximately 10 g of the prothrombin complexconcentrate being employed for a 20 ml monoclonal antibody column.Subsequently, the dissolved prothrombin complex concentrate wasfiltered, centrifuged at 20,000 r.p.m. for 15 min and sterilely filteredthrough a 0.8 μm filter. The sterilely filtered and dissolvedprothrombin complex concentrate was applied to the column at a flow rateof 10 ml/h. Subsequently, the column was washed free of protein with thewashing buffer, and finally the bound protein C was eluted by means ofthe elution buffer at a flow rate of 5 ml/h and the fractions werecollected. The eluted protein C was dialyzed against a buffer (0.2 mol/lTris, 0.15 M glycine and 1 mM EDTA, pH 8.3). Protein C antigenconcentration was determined using the method described by C. B.Laurell, Scand. J. Clin. Lab. Invest. 29, Suppl. 124:21-37 (1972), andprotein C activity was determined using Protac activation.

The protein C eluate obtained was finished to a pharmaceuticallyapplicable preparation in the following manner:

The eluate was first subjected to ultrafiltration and diafiltrationsteps. Diafiltration was carried out with a buffer containing 150 mmolNaCl and 15 mmol trisodium citrate.2H₂O per liter, at a pH of 7.4. Theobtained filtrate was freeze-dried and virus inactivated by a one-hourvapor treatment at 80° C.±5° C. and at 1375±35 mbar.

The lyophilized, virus inactivated material was then dissolved in asterile isotonic NaCl solution and potentially present antibodies orserum amyloid P were eliminated by means of ion exchange chromatographyon Q-Sepharose. The purified solution was concentrated by means of anadditional ultrafiltration and diafiltration step. After this step, 10 galbumin, 150 mmol NaCl and 15 mmol trisodium citrate per liter wereadded to the solution obtained. The pH of the solution was 7.5. Neithermurine immunoglobulin nor factors II, VII, IX and X could be detected.Subsequently, the solution was sterilely filtered, filled in containersand lyophilized. The specific activity was 14 units protein C per mg ofprotein. One unit of protein C activity is defined as the protein Cactivity in 1 ml normal plasma and is calibrated against the firstinternational standard of protein C. An amidolytic assay was used as theactivity test, wherein protein C was activated by means of Protac(Pentapharm), a common protein C activator produced from a snake venompreparation.

Time-dependent Degradation of Protein C

Protein C was treated with plasmin and the degradation was observed bymeans of immunoblotting. To this end, 270 μl of a protein C-containingsolution (8 μg/ml) were incubated with 270 μl plasmin (1 CU/ml) at 37°C. Accordingly, the substrate/enzyme ratio was 8:1 (μg/CU). After only60 minutes, no protein C could be amidolytically detected any longer.

Dose-dependent Degradation of Protein C

In order to investigate the dose-dependent degradation of plasmaticprotein C, 50 μl plasmin were each added to 50 μl human citrated plasmaat concentrations of 10, 5, 3, 1.5 and 0.5 CU/ml, respectively. After areaction time of 10 minutes, 50 μl antithrombin III-heparin complex (10U ATIII, 50 U heparin per ml) were each added. By this addition, thereaction is stopped.

Protein C was amidolytically determined with the specific chromogenicsubstrate S 2366 (Kabi) upon activation with PROTAC (Pentapharm).

For comparison, plasmatic protein C without plasmin addition was treatedin parallel. The results are apparent from the Figure (abscissa: CUplasmin; ordinate: % protein C activity). The Figure illustrates thatprotein C is completely degraded after only 10 minutes if a solutioncontaining 10 CU/ml plasmin has been added.

What we claim is:
 1. A pharmaceutical preparation comprising protein Cand a thrombolytically active substance that does not activate proteinC.
 2. A pharmaceutical preparation as set forth in claim 1, wherein saidthrombolytically active substance is selected from the group consistingof urokinase, tPA, Lys-plasminogen and streptokinase.
 3. A method oftreating thrombosis and preventing reocclusion in a patient comprisingthe step of administering an effective amount of protein C and athrombolytically active substance unable to directly activate protein C.4. A method of preventing reocclusion in a patient during thrombolysistherapy comprising the administration of an effective amount of proteinC prior to, during and/or after the thrombolysis therapy.
 5. A methodfor producing a drug to treat thrombosis and prevent reocclusion,comprising the step of combining protein C with a thrombolyticallyactive substance unable to activate protein C.
 6. A method according toclaim 3, wherein 30 to 80 U/kg of protein C are administered to saidpatient 2-3 times per day.
 7. A method according to claim 3, wherein 15to 30 U/kg per hour of protein C are administered to said patient.